Heterotrimeric G proteins are widely thought to play a critical role in regulating synaptic strength, yet relatively little is known about how these signaling pathways regulate synaptic vesicle exocytosis and recycling. We identified a muscarinic acetylcholine (ACh) receptor (GAR-2) and a GABAe receptor (GBB- 1/2) that are expressed in cholinergic motor neurons, and which inhibit acetylcholine (ACh) release at neuromuscular junctions (NMJs). We showed that presynaptic diaclyglycerol (DAG), produced by G protein signaling pathways, stimulate ACh secretion. We identified two DAG/phorbol ester targets, UNC-13 and protein kinase C (kin-13 PKC), that mediate these effects. We identify tomosyn as an inhibitor of ACh release. And we identify 175 proteins required for ACh release in a systematic RNAi screen. Here, we propose three new aims to further investigate mechanisms regulating synaptic vesicle exo- and endocytosis. First, we will test two alternative models for UNC-13 function in exocytosis. Does UNC-13 promote formation of open syntaxin or does UNC-13 prevent formation of inhibitory tomosyn.tSNARE complexes? Second, we will characterize the function of UNC-18 in anterograde transport of syntaxin, and possibly other active zone proteins. Third, we will analyze endocytic and synapse formation defects caused by mutations in four genes identified in the RNAi screen and we will continue characterizing the set of 175 genes identified in the RNAi screen by functional and fluorescence profiling. In summary, changes in the G protein signaling have been proposed to regulate many aspects of synaptic transmission yet much remains to be learned about the biochemical pathways leading to these changes. The experiments proposed here should give significant new insights into the mechanisms of synaptic vesicle priming and recycling, and about how these processes are regulated by G protein cascades. Given the strong phylogenetic conservation of these pathways, it is likely that our experiments will also shed light on these aspects of synaptic cell biology in other organisms.